CN1425911A - Raman test method for quick eliminating biologichal molecular fluorescence - Google Patents
Raman test method for quick eliminating biologichal molecular fluorescence Download PDFInfo
- Publication number
- CN1425911A CN1425911A CN03112706.1A CN03112706A CN1425911A CN 1425911 A CN1425911 A CN 1425911A CN 03112706 A CN03112706 A CN 03112706A CN 1425911 A CN1425911 A CN 1425911A
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- raman
- nitrobenzene
- fluorescence
- testing
- centrifugal
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- 238000001069 Raman spectroscopy Methods 0.000 title claims abstract description 23
- 238000010998 test method Methods 0.000 title claims abstract description 6
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000012360 testing method Methods 0.000 claims abstract description 16
- 238000001237 Raman spectrum Methods 0.000 claims description 12
- 239000012472 biological sample Substances 0.000 claims description 4
- 238000001228 spectrum Methods 0.000 claims description 4
- 230000008030 elimination Effects 0.000 claims description 2
- 238000003379 elimination reaction Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 6
- 238000010791 quenching Methods 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000010586 diagram Methods 0.000 abstract 1
- 230000003595 spectral effect Effects 0.000 abstract 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 229940098773 bovine serum albumin Drugs 0.000 description 8
- 102000006382 Ribonucleases Human genes 0.000 description 7
- 108010083644 Ribonucleases Proteins 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000843 powder Substances 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000283730 Bos primigenius Species 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present inventinon provides a kind of Raman test method for quick eliminating biological molecular fluorescence. Nitrobenzene in very small amount is first mixed into tested biological molecular solution, the solution is set in centrifugal test tube, and after centrifugal analysis in centrifugal machine nitrobenzene is eliminated. The said method can quench fluorescence effectively to obtain clear and stable Raman spectral diagram quickly and has few influence on the conformation of biological molecular image.
Description
Technical field
The present invention relates to a kind of method of when carrying out the Raman test, eliminating biomolecule fluorescence fast, belong to the technical field of Raman spectrum test.
Background technology
At present, the laser raman technology is used to survey the molecular structure of protein and other more and more frequently, particularly their native conformations in the low concentration aqueous solution.But most protein fluorescence can occur under laser radiation, and fluorescence needs only appearance, and its intensity always surpasses Raman signal, so that be difficult to obtain clear and legible Raman collection of illustrative plates.For a long time, in order to eliminate fluorescence, mainly the method that all adopts high power laser that specimen is carried out the long period irradiation makes fluorescence intensity degenerate to acceptable level.This irradiation, along with the difference of sample, the time that needs is also different, and what have only needs dozens of minutes, and what have but reaches tens of hours and does not also reach desirable effect.Even eliminate the pre-service of fluorescence through laser radiation, when test protein etc. has the biological sample laser Raman spectroscopy of big fluorescent yield, for preventing fluorescence interference and fluctuation, necessary sweep velocity is very slow, integral time is very long, as in the sweep limit of characterising biological molecular characterization commonly used, from 500cm
-1To 1800cm
-1, often run-down needs a few hours even more than tens of hours.
Summary of the invention
The present invention is exactly the Raman method of testing to a kind of quick elimination biomolecule fluorescence of the deficiency proposition of existing method, the method is quench fluorescence effectively, obtain steady and audible Raman spectrogram rapidly, and very little to the conformation influence of biomolecule such as protein itself.
The technical solution adopted for the present invention to solve the technical problems is: earlier the pure nitrobenzene of few quantitative analysis is mixed the biomolecule solution that will test, stir slightly, put in the centrifuge tube, again with after the centrifugal layering of hydro-extractor nitrobenzene being removed.Handle back fluorescence so then substantially by quencher, can test the steady and audible laser raman spectrum of the biomolecule that has the nitrobenzene background rapidly.Test the laser raman spectrum of nitrobenzene more separately with similarity condition, with expression NO
2The 1350cm of symmetrical stretching vibration
-1Be reference peaks, carry out the baseline correction of spectrum peak with computing machine, the background of deduction nitrobenzene Raman spectrum so just can obtain goodish this biomolecule solution Raman spectrogram of having removed fluorescence.
Embodiment
1. the ribonuclease crystalline powder is dissolved in the redistilled water, be mixed with 4% Ribonuclease in Aqueous Solution, add the nitrobenzene that accounts for Ribonuclease in Aqueous Solution total amount 1%, stirred 3 minutes with stirrer, put in the centrifuge tube, through supercentrifuge layering after centrifugal 15 minutes, take out the limpid Ribonuclease in Aqueous Solution testing laser Raman spectrum in upper strata.
After above-mentioned processing, the fluorescence of protein molecule is almost entirely by quencher.With 200mW shoot laser power and 2cm
-1/ sec sweep velocity, the Raman spectrogram of gained is steady and audible.Scan altogether 8 times, owing to overcome fluorescence interference, the speed that obtains Raman spectrogram is accelerated greatly, as long as minute just can scan one time surplus in the of ten.By computing machine acquired signal is made progressive mean again.With the laser raman spectrum of similarity condition test nitrobenzene, with expression NO
2The 1350cm of symmetrical stretching vibration
-1Be reference peaks, carry out the baseline correction of spectrum peak with computing machine, the background of deduction nitrobenzene Raman spectrum, what obtain like this is quite successful Ribonuclease in Aqueous Solution Raman spectrogram.This spectrogram with delivered the document basically identical abroad, and qualitative analysis goes out the secondary structure of ribonuclease thus, the conformation of former ribonuclease is not affected.
With the bovine serum albumin(BSA) powder dissolution in redistilled water, be mixed with 5% bovine serum albumin solution, add the nitrobenzene that accounts for bovine serum albumin solution total amount 0.5%, stirred 2 minutes with stirrer, put in the centrifuge tube, through supercentrifuge layering after centrifugal 20 minutes, take out the limpid bovine serum albumin solution testing laser Raman spectrum in upper strata.
After above-mentioned processing, the fluorescence of bovine serum albumin(BSA) molecule is almost entirely by quencher.With 200mW shoot laser power and 1cm
-1/ sec sweep velocity, the Raman spectrogram of gained is steady and audible.Scan altogether 6 times, owing to overcome fluorescence interference, the speed that obtains the bovine serum albumin(BSA) Raman spectrogram is accelerated greatly, as long as minute just can scan one time surplus in the of 20.By computing machine the signal of gathering is made progressive mean again.With the laser raman spectrum of similarity condition test nitrobenzene, with expression NO
2The 1350cm of symmetrical stretching vibration
-1Be reference peaks, carry out the baseline correction of spectrum peak with computing machine, the background of deduction nitrobenzene Raman spectrum so just obtains quite successful bovine serum albumin solution Raman spectrogram.This spectrogram with delivered the document basically identical abroad, and qualitative analysis goes out the secondary structure of bovine serum albumin(BSA) thus, the sero-abluminous conformation of aurochs is not affected.This method is of practical significance to the Raman test of biomolecule very much as can be known.
Claims (2)
1. Raman method of testing of eliminating fast biomolecule fluorescence, it is characterized in that the nitrobenzene that will account for solution total amount 0.1%-2% earlier mixes the biological sample solution that will test, stirred 1-10 minute, put in the centrifuge tube, after using the centrifugal 15-30 of hydro-extractor minute layering again, take out the limpid biological sample solution testing laser Raman spectroscopy in upper strata.
2. the Raman method of testing of a kind of quick elimination biomolecule fluorescence according to claim 1 is characterized in that with behind the above-mentioned condition test biological sample solution laser Raman spectroscopy, tests the laser raman spectrum of nitrobenzene more separately with similarity condition, with expression NO
2The 1350cm of symmetrical stretching vibration
-1Be reference peaks, carry out the baseline correction of spectrum peak, the background of deduction nitrobenzene Raman spectrum with computing machine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03112706.1A CN1425911A (en) | 2003-01-17 | 2003-01-17 | Raman test method for quick eliminating biologichal molecular fluorescence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03112706.1A CN1425911A (en) | 2003-01-17 | 2003-01-17 | Raman test method for quick eliminating biologichal molecular fluorescence |
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| Publication Number | Publication Date |
|---|---|
| CN1425911A true CN1425911A (en) | 2003-06-25 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN03112706.1A Pending CN1425911A (en) | 2003-01-17 | 2003-01-17 | Raman test method for quick eliminating biologichal molecular fluorescence |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103411953B (en) * | 2013-08-19 | 2015-11-18 | 东莞市华立实业股份有限公司 | A kind of method of farm chemical emulsion preparation being carried out to field quick detection |
| CN105466908A (en) * | 2015-12-31 | 2016-04-06 | 安徽芯核防务装备技术股份有限公司 | Raman spectrum method for removing interference noise produced during sample bottle fixing |
| CN105606589A (en) * | 2016-02-18 | 2016-05-25 | 广西科技大学 | Raman spectrum fluorescence eliminating method for judging and obtaining fluorescence fading measured value through kurtosis |
| CN105699356A (en) * | 2016-02-19 | 2016-06-22 | 广西科技大学 | Method for judging fluorescence quenching degree of Raman spectrum through information entropy |
| CN107957414A (en) * | 2017-10-27 | 2018-04-24 | 华南理工大学 | A kind of method with potassium bromide quenching fluorescence in Raman spectrum |
-
2003
- 2003-01-17 CN CN03112706.1A patent/CN1425911A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103411953B (en) * | 2013-08-19 | 2015-11-18 | 东莞市华立实业股份有限公司 | A kind of method of farm chemical emulsion preparation being carried out to field quick detection |
| CN105466908A (en) * | 2015-12-31 | 2016-04-06 | 安徽芯核防务装备技术股份有限公司 | Raman spectrum method for removing interference noise produced during sample bottle fixing |
| CN105466908B (en) * | 2015-12-31 | 2018-04-20 | 安徽芯核防务装备技术股份有限公司 | A kind of sample bottle fixes the Raman spectrum minimizing technology of interference noise |
| CN105606589A (en) * | 2016-02-18 | 2016-05-25 | 广西科技大学 | Raman spectrum fluorescence eliminating method for judging and obtaining fluorescence fading measured value through kurtosis |
| CN105699356A (en) * | 2016-02-19 | 2016-06-22 | 广西科技大学 | Method for judging fluorescence quenching degree of Raman spectrum through information entropy |
| CN105699356B (en) * | 2016-02-19 | 2018-10-19 | 广西科技大学 | Judge the method that the fluorescence of Raman spectrum eliminates degree by comentropy |
| CN107957414A (en) * | 2017-10-27 | 2018-04-24 | 华南理工大学 | A kind of method with potassium bromide quenching fluorescence in Raman spectrum |
| CN107957414B (en) * | 2017-10-27 | 2020-09-22 | 华南理工大学 | Method for quenching fluorescence by using potassium bromide in Raman spectrum |
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